Contact PD/PI: Wang, Hong 1 Sister chromatid cohesion is mediated by the cohesin complex, which also plays key roles in diverse biological 2 pathways including DNA replication restart, and DNA double-strand break (DSB) repair under genotoxic stress. 3 In vertebrates, the core cohesin complex consists of a tripartite ring assembled from SMC1, SMC3, RAD21, 4 and the fourth subunit SA1 or SA2. Germline mutations in core cohesin subunits lead to a wide spectrum of 5 human diseases collectively called ?cohesinopathies, and to increased incidence of cancers. Our current 6 understanding of cohesin functions is based on models in which cohesin DNA binding is mediated exclusively 7 through nonspecific entrapment of dsDNA by the cohesin ring. However, these models cannot explain 8 observations from numerous studies demonstrating the localization of the cohesin complex at specific 9 structures and sequences along the genome. We recently published a novel observation from single-molecule 10 studies demonstrating that SA1 binds specifically to double-stranded telomeric DNA. In addition, we found that 11 SA2 displays high binding specificities for ssDNA gaps. These new results raise fundamental questions 12 regarding the structure and dynamics of SA1/2-DNA complexes and their roles in preserving genome integrity. 13 We hypothesize that DNA sequence and structure-dependent DNA binding by cohesin SA1/2 preserve 14 genome integrity under genotoxic stress. The proposed work tests this hypothesis in the following ways. 15 First, using both bulk assays and single-molecule atomic force and real-time fluorescence microscopy imaging 16 of proteins on DNA, we will identify the domains on SA1/2 that mediate DNA binding, and the impact of 17 nucleosomes on their DNA binding dynamics. Second, the mechanism underlying the unique cohesin-ring 18 independent SA1-mediated sister telomere cohesion process is still unknown. We will test the ?multi-site SA1- 19 TRF1-TIN2-mediated DNA-DNA bridging? model for sister telomere cohesion. We will define the architecture of 20 the telomeric cohesin complex by using a novel electrostatic force microscopy (EFM) imaging technique to 21 reveal DNA paths within the assembled protein structure. To study the sequential steps in protein recruitment 22 and DNA-DNA bridging mediated by SA1-TRF1-TIN2 protein complexes, we will use real-time fluorescence 23 microscopy imaging of DNA molecules confined to nanochannels in microfluidic devices. Finally, to determine 24 roles for SA1/2 in preventing DNA damage-induced genomic and telomeric instability, we will investigate the 25 impact of SA1/2 DNA binding mutants and protein depletion on the efficiency of joining distal DNA ends during 26 DNA DSB repair in cultured cells. Furthermore, we hypothesize that SA1-mediated telomere cohesion prevents 27 DNA damage-induced telomere instability. To explore this postulate, we will examine how SA1 DNA binding 28 mutants and protein depletion impact telomere integrity after cellular exposure to genotoxic agents that 29 damage telomeres. These results will greatly advance our understanding of the cohesin function in diverse 30 genome maintenance pathways. 31 32 33 34 35 36 37 38 39 40 Project Summary/Abstract Page 7